Leptin directly activates SF1 neurons in the VMH, and this action by leptin is required for normal body-weight homeostasis

Leptin, an adipocyte-derived hormone, acts directly on the brain to control food intake and energy expenditure. An important question is the identity of first-order neurons initiating leptin's anti-obesity effects. A widely held view is that most, if not all, of leptin's effects are mediated by neurons located in the arcuate nucleus of the hypothalamus. However, leptin receptors (LEPRs) are expressed in other sites as well, including the ventromedial hypothalamus (VMH). The possible role of leptin acting in "nonarcuate" sites has largely been ignored. In the present study, we show that leptin depolarizes and increases the firing rate of steroidogenic factor-1 (SF1)-positive neurons in the VMH. We also show, by generating mice that lack LEPRs on SF1-positive neurons, that leptin action at this site plays an important role in reducing body weight and, of note, in resisting diet-induced obesity. These results reveal a critical role for leptin action on VMH neurons.     

Divergence genetics analysis reveals historical population genetic processes leading to contrasting phylogeographic patterns in co-distributed species

Coalescent samplers are computational time machines for inferring the historical demographic genetic processes that have given rise to observable patterns of spatial genetic variation among contemporary populations. We have used traditional characterizations of population structure and coalescent‐based inferences about demographic processes to reconstruct the population histories of two co‐distributed marine species, the frilled dog whelk, Nucella lamellosa, and the bat star, Patiria miniata. Analyses of population structure were consistent with previous work in both species except that additional samples of N. lamellosa showed a larger regional genetic break on Vancouver Island (VI) rather than between the southern Alexander Archipelago as in P. miniata. Our understanding of the causes, rather than just the patterns, of spatial genetic variation was dramatically improved by coalescent analyses that emphasized variation in population divergence times. Overall, gene flow was greater in bat stars (planktonic development) than snails (benthic development) but spatially homogeneous within species. In both species, these large phylogeographic breaks corresponded to relatively ancient divergence times between populations rather than regionally restricted gene flow. Although only N. lamellosa shows a large break on VI, population separation times on VI are congruent between species, suggesting a similar response to late Pleistocene ice sheet expansion. The absence of a phylogeographic break in P. miniata on VI can be attributed to greater gene flow and larger effective population size in this species. Such insights put the relative significance of gene flow into a more comprehensive historical biogeographic context and have important implications for conservation and landscape genetic studies that emphasize the role of contemporary gene flow and connectivity in shaping patterns of population differentiation.     

Dimethylthiourea protects against chlorine induced changes in airway function in a murine model of irritant induced asthma

Background

Exposure to chlorine (Cl₂) causes airway injury, characterized by oxidative damage, an influx of inflammatory cells and airway hyperresponsiveness. We hypothesized that Cl₂-induced airway injury may be attenuated by antioxidant treatment, even after the initial injury.

Methods

Balb/C mice were exposed to Cl₂ gas (100 ppm) for 5 mins, an exposure that was established to alter airway function with minimal histological disruption of the epithelium. Twenty-four hours after exposure to Cl₂, airway responsiveness to aerosolized methacholine (MCh) was measured. Bronchoalveolar lavage (BAL) was performed to determine inflammatory cell profiles, total protein, and glutathione levels. Dimethylthiourea (DMTU;100 mg/kg) was administered one hour before or one hour following Cl₂ exposure.

Results

Mice exposed to Cl₂ had airway hyperresponsiveness to MCh compared to control animals pre-treated and post-treated with DMTU. Total cell counts in BAL fluid were elevated by Cl₂ exposure and were not affected by DMTU treatment. However, DMTU-treated mice had lower protein levels in the BAL than the Cl₂-only treated animals. 4-Hydroxynonenal analysis showed that DMTU given pre- or post-Cl₂ prevented lipid peroxidation in the lung. Following Cl₂ exposure glutathione (GSH) was elevated immediately following exposure both in BAL cells and in fluid and this change was prevented by DMTU. GSSG was depleted in Cl₂ exposed mice at later time points. However, the GSH/GSSG ratio remained high in chlorine exposed mice, an effect attenuated by DMTU.

Conclusion

Our data show that the anti-oxidant DMTU is effective in attenuating Cl₂ induced increase in airway responsiveness, inflammation and biomarkers of oxidative stress.     

Intersection of endocytic and exocytic systems in Toxoplasma gondii

Host cytosolic proteins are endocytosed by Toxoplasma gondii and degraded in its lysosome‐like compartment, the vacuolar compartment (VAC), but the dynamics and route of endocytic trafficking remain undefined. Conserved endocytic components and plant‐like features suggest T. gondii endocytic trafficking involves transit through early and late endosome‐like compartments (ELCs) and potentially the trans‐Golgi network (TGN) as in plants. However, exocytic trafficking to regulated secretory organelles, micronemes and rhoptries, also proceeds through ELCs and requires classical endocytic components, including a dynamin‐related protein, DrpB. Here, we show that host cytosolic proteins are endocytosed within 7 minutes post‐invasion, trafficked through ELCs en route to the VAC, and degraded within 30 minutes. We could not definitively interpret if ingested protein is trafficked through the TGN. We also found that parasites ingest material from the host cytosol throughout the parasite cell cycle. Ingested host proteins colocalize with immature microneme proteins, proM2AP and proMIC5, in transit to the micronemes, but not with the immature rhoptry protein proRON4, indicating that endocytic trafficking of ingested protein intersects with exocytic trafficking of microneme proteins. Finally, we show that conditional expression of a DrpB dominant negative mutant increases T. gondii ingestion of host‐derived proteins, suggesting that DrpB is not required for parasite endocytosis.      

NmeCas9 is an intrinsically high-fidelity genome-editing platform

Background

The development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wildtype SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs are considerably smaller and therefore better suited for viral delivery.

Results

Here we show that wildtype NmeCas9, when programmed with guide sequences of the natural length of 24 nucleotides, exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5′-N₄GATT-3′), for NmeCas9 genome editing in human cells.

Conclusions

Our results show that NmeCas9 is a naturally high-fidelity genome-editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering.

     

GROWTH, MORTALITY AND FECUNDITY IN SUCCESSIVE GENERATIONS OF HEIX ASERSA MULLER CULTURED INDOORS AND CROWDING EFFECTS ON FAST-, MEDIUM- AND SLOW-GROWING SNAILS OF THE SAME CLUTCH

This paper examines the optimum conditions for edible snailsHelix aspersa to be cultured indoors successfully in successivegenerations (originating from the crossing of snails comingfrom different clutches of a previous generation), and the effectof crowding on growth and reproduction in fast-, medium-, andslow-growing snails coming from the same clutches. The timeneeded for the snails to reach marketable size (25–32mm)varied from 2.5 to 5 months up to the 7th generation. The timeneeded for the snails to mature and reproduce from 4 to 7 monthsuntil the fifth generation. After the F5 x F5 generation, thefinal size of the snails decreased. The number of eggs did notdiffer statistically among the different generations but thereproductive success (how many snails reproduced/cage) increasedfrom Fl = Fl generation onwards to F5 x F5. In F6 x F6 onlythree (out of 26) snails reproduced and in F7 x F7 none, althoughthe snails remained under controlled conditions for 15 moremonths. Mortality in the different generations varied from 0–10%up to F5 x F5 but from F6 x F6 onwards increased and reached25%. Concerning the origin of snails, it was found that largersnails (originating from Kyparissia, Peloponnesos) lay statisticallymore eggs (138.40 ± 29.60, N =5) than smaller ones (77.38± 40.42, N=4) (originating from Hania, island of Crete).Hatching success was greater, too. (Received 10 September 1996; accepted 24 March 1997)     

Time course of airway remodelling after an acute chlorine gas exposure in mice

Accidental chlorine (Cl₂) gas inhalation is a common cause of acute airway injury. However, little is known about the kinetics of airway injury and repair after Cl₂ exposure. We investigated the time course of airway epithelial damage and repair in mice after a single exposure to a high concentration of Cl₂ gas. Mice were exposed to 800 ppm Cl₂ gas for 5 minutes and studied from 12 hrs to 10 days post-exposure. The acute injury phase after Cl₂ exposure (≤ 24 hrs post-exposure) was characterized by airway epithelial cell apoptosis (increased TUNEL staining) and sloughing, elevated protein in bronchoalveolar lavage fluid, and a modest increase in airway responses to methacholine. The repair phase after Cl₂ exposure was characterized by increased airway epithelial cell proliferation, measured by immunoreactive proliferating cell nuclear antigen (PCNA), with maximal proliferation occurring 5 days after Cl₂ exposure. At 10 days after Cl₂ exposure the airway smooth muscle mass was increased relative to controls, suggestive of airway smooth muscle hyperplasia and there was evidence of airway fibrosis. No increase in goblet cells occurred at any time point. We conclude that a single exposure of mice to Cl₂ gas causes acute changes in lung function, including pulmonary responsiveness to methacholine challenge, associated with airway damage, followed by subsequent repair and airway remodelling.